3 research outputs found
The molecular recognition of epothilones by microtubules and tubulin dimers revealed by biochemical and NMR approaches
36 p.-6 fig.+ 9 p.-2 fig.-4 tab. (support. inf.)The binding of epothilones to dimeric tubulin and to microtubules has been studied
by means of biochemical and NMR techniques. We have determined the binding constants of epothilone A (EpoA) and B (EpoB) to dimeric tubulin, which are four orders of magnitude lower than those for microtubules, and we have elucidated the conformation and binding epitopes of EpoA and EpoB when bound to tubulin dimers and microtubules in solution.
The determined conformation of epothilones when bound to dimeric tubulin is similar to that found by X-ray crystallographic techniques for the binding of EpoA to the
Tubulin/RB3/TTL complex; it is markedly different from that reported for EpoA bound to
zinc-induced sheets obtained by electron crystallography. Likewise, only the X-ray
structure of EpoA bound to the Tubulin/RB3/TTL complex at the luminal site, but not the
electron crystallography structure, is compatible with the results obtained by STD on the
binding epitope of EpoA bound to dimeric tubulin, thus confirming that the allosteric
change (structuring of the M-loop) is the biochemical mechanism of induction of tubulin
assembly by epothilones. TR-NOESY signals of EpoA bound to microtubules have been
obtained supporting the interaction with a transient binding site with a fast exchange rate
(pore site), consistent with the notion that epothilones access the luminal site through the
pore site, as has also been observed for taxanes. Finally, the differences in the tubulin
binding affinities of a series of epothilone analogs has been quantitatively explained using the newly determined binding pose and the COMBINE methodology.This research was supported by BIPPED2 (S2010/BMDâ2457)
(to J.F.DĂaz and F.Gago), and MHit (CAM S2010/BMD-2353 to J.JĂmenez-Barbero,J.M.Andreu) projects of the Comunidad de Madrid, the BIO2010â16351 (to J.F.DĂaz) and CTQ2012-32025 (to J.JĂmenez-Barbero) and SAF2009-13914-C02-02 (to F. Gago) projects from the Ministry of Economy and Competitiveness of Spain, and a Ramon y Cajal grant to A. Canales.Peer reviewe